Antigens

ABSTRACT

Antigens associated with carcinomas and adenocarcinomas as well as methods for isolating, identifying and detecting them are disclosed.

United States Patent [15] 3,697,638 Hansen [45] Oct. 10, 1972 ANTIGENS[56] References Cited Inventor: Hans John Hansen, Allendale, [73]Asslgnee gg Nutley Thomson et al., Proc. Natl. Acad. Sci. u.s., 64:161-

167 (1969) The Radioimmunoassay of Circulating [22] Filed: April 12,1971 CEA of the Human Digestiye System. 21 A l. N 1 1 pp 0 33 404Primary ExaminerLeland A. Sebastian Related US. Application DataAssistant Examiner-S. R. Hellman Att0meySamuel L. Welt Jon S. SaxeBernard S. [63] Continuation-impart of Ser. No. 110,288, Jan.

27, 1971, which is a continuation-in-part of if? Gerald Rosen Ham Swopeand wllham Ser. No. 42,526, June 1, 1970. are) 52 us. Cl. ..424/1,424/12, 424/88, [57] ABSTRACT 23/230B Antigens associated withcarcinomas and adenocarlnt- Cl- ..A61k inema as well as methods forisolating [58] Field of Search....424/l, 8, 12, 88; 252/301.l R, anddetecting them are i l 20 Claims, N0 Drawings ANTIGENS RELATEDAPPLICATIONS This is a continuation-impart of US. Pat. Application Ser.No. 110,288, filed Jan. 27, I971 which in turn is a continuation-in-partof US. Pat. Application Ser. No. 42,526, filed June 1, l970.

BACKGROUND OF THE INVENTION The neoplastic process in human beings hasbeen and still is the subject of intensive study. In order to obtain abetter understanding of the disease, human cancer tissue has beenstudied in an effort to discover the cause, treatment, prevention and/ordiagnosis of cancer. Early diagnosis of cancer is very important sinceit increases the chances of effecting a complete remission of thedisease.

In an effort to utilize known diagnostic tools to detect the presence ofcancer tumors, attempts have been made to demonstrate tumor specificantigens to human carcinomas. These attempts have previously beenunsuccessful with many types of carcinomas since it has not beenpossible to segregate normal tissue antigens from abnormalcancerantigens and demonstrate the specificity of the cancer antigens.

In the efforts to isolate abnormal cancer antigens and demonstrate theirspecificity, attempts have been made to cause the formation of tumorspecific antibodies and demonstrate their presence in sera obtained fromanimals immunized with preparationsof human cancer. If consistentlyreproducible, the demonstration of the presence of tumor-specificantibodies in animal antisera would lead to the use of a valuablediagnostic tool.

In order to fully utilize the existence of tumorspecific antibodies inanimal antisera as a diagnostic tool, a test must be developed whichwill demonstrate the presence of the tumor antigen in the blood of thepatient. Procedures which have been devised have not proven efficient orsensitive in the detection of and differentiation between carcinomasoriginating at different locations within the body, or metastaticconditions.

Among the possible sources of antigens associated with human carcinomaswhich have been most extensively studied by investigators areadenocarcinoma of the colon and digestive tract, meconium, carcinoma ofthe liver, ovarian cysts and carcinoma of the breast. Sinceadenocarcinoma of the colon is one of the most widespread cancers andusually requires a surgical procedure for definitive diagnosis, aftersome gross symptomatology has developed, it has been among the mostextensively studied.

Efforts to extract a relatively pure antigen associated with carcinomasor adenocarcinomas have met with either no success or are impracticalfrom a commercial point of view since a process has not been found tomake it possible to completely segregate such an antigen from normaltissue antigens and non-antigenic materials.

The presence of antigens which are stated to be specific toadenocarcinomas of the colon and digestive system by means ofimmunological tolerance and absorption techniques have beendemonstrated, Gold et al., J. Expt. Med. 121 439462 (1965). However, thepractical and commercially feasible isolation of the antigen itself aswell as its association with carcinomas and adenocarcinomas had, untilthe present invention, not been achieved.

The tumor-specific antigens have been previously shown to be presentonly in patients who have adenocarcinoma which originate in digestivesystem epithelium derived from embryonic entodermal tissue, i.e.,esophagus, stomach, duodenom, pancreas and rectum.

It has also previously been demonstrated that the tumor-specific antigenis also present in the digestive organs of fetuses between 2 and 6months of gestation, Gold et al., J. Exptl. Med. 122 467-487 (1965).Thus, for convenience, the antigen has been designed as carcinoembryonicantigen (CEA).

Not only has it heretofore not been possible to isolate and characterizethe CEA by practical rapid methods, but it has not been possible todemonstrate its presence in the blood of persons having adenocarcinomawith a diagnostic test suitable for large screening programs.

DETAILED DESCRIPTION OF THE INVENTION I have discovered that thematerial heretofore known as carcinoembryonic antigen (CEA) is a mixtureof several components, at least two of which have antigenic activitywhich is associated with human carcinoma generally. These two activecomponents are called carcinoembryonic antigen component A andcarcinoembryonic antigen component B.

This invention in one significant aspect relates to a method offractionating material having carcinoembryonic antigen activity into itscomponent parts, e.g., carcinoembryonic antigen component A andcarcinoembryonic antigen component B.

This invention in further aspects relates to methods of isolating andcharacterizing the carcinoembryonic antigen components associated withcarcinomas, for diagnostic test procedures and for utilizing eitherradioactive tagged carcinoembryonic antigen material, component A orcomponent B to detect circulating carcinoembryonic antigen material,component A and/or component B.

This invention also relates to a diagnostic test method useful in thedetection of carcinoma and suitable for post-operative monitoring ofcarcinoma patients. Carcinoma, as used herein, includes all carcinomasand adenocarcinomas present in humans. As used herein, carcinoembryonicantigen material, means the material with carcinoembryonic antigenactivity which contains component A and/or component B.

In order to produce radioactive tagged carcinoembryonic antigenmaterial, component A or component B individually and utilize eachseparately in the improved diagnostic tests of this invention, it isfirst necessary to isolate and purify each entity and confirm itsidentity by means of specific anti-bodies.

According to this invention, practical processes have been discoveredfor:

a. Isolating, purifying, characterizing and confirming the identity andspecificity of carcinoembryonic antigen material, component A andcomponent B;

b. Utilizing radioactive tagged carcinoembryonic antigen material,component A and/or component B to detect the presence of carcinoma bythe detection of circulating antigens; and

c. Differentiating between circulating free and total carcinoembryonicantigen material, component A and/or component B.

While this invention is concerned with antigens associated withcarcinomas generally the isolation and purification procedures describedherein will refer to colon carcinoma tissue and meconium which arerepresentative of the materials containing CEA material, component Aand/or component B.

Material having carcinoembryonic antigen activity is isolated andpurified according to the process of this invention by homogenizingadenocarcinoma tissue from primary or metastatic tumors, preferablythose originating within the digestive system, with tumors from thecolon, for example, being suitable, or by homogenizing meconium.

In order to isolate the carcinoembryonic antigen material, component Aand/or component B associated with the homogenized material, it isnecessary to first separate all other material from the homogenate,isolate the carcinoembryonic antigen material, then isolate theindividual components of the carcinoembryonic antigen material thereof.This is accomplished by chemical and physical extraction andpurification procedures. If, for example, only component A or componentB are present in the homogenate, then the isolation and purificationprocedures will produce the component without fractionation.

Once the extraction and purification procedures are completed, theidentity of the finally isolated fractions as carcinoembryonic antigenmaterial or a component must be confirmed. This can be accomplished byvarious known techniques, e.g., double diffusion in agar gel,immuno-electrophoresis, hemagglutination, passive cutaneous anaphylaxisand the like.

In order to utilize these techniques, the antibodies used must beconfirmed to be specific for the CEA material, component A and/orcomponent B. Antibodies which meet this criteria can be produced byimmunological tolerance or absorption techniques.

In the absorption technique, tumor antiserum is absorbed with normaltissue and normal fluids (saliva, serum, plasma) in order to removeanti-normal components of the antiserum. Any residual antibody activityin the absorbed antiserum which is directed against tumor material isthen considered to be tumor specific. This method is not without itsfaults since there is the possibility that tumor specific antibodies mayhave been removed or inactivated by normal tissue components similar to,but not identical with, the tumor antigens which initially stimulatedthe antibody production.

In the immunological tolerance technique, animals are renderedimmunologically tolerant to normal tissues during neonatal life. Thetolerant animals are then immunized with tumor preparations of the samedonor species. Where adequate suppression of the immune response tonormal tissue components has been achieved, the development ofantibodies apparently specific for the carcinoembryonic antigen activityhas been achieved.

Colon adenocarcinoma tumor tissue and normal colon tissue from the sameindividual can be utilized to illustrate this technique becauseadenocarcinoma of the colon almost never extends submucosally more than6 to 7 cm. on either side of a tumor visible in the gross.

The colon adenocarcinoma tumor tissue and normal colon tissue from thesame individual are treated separately but in parallel fashion. Thetissue is ground up, suspended in a buffer, then homogenized. Thehomogenate is then treated to remove solid particles. Centrifugation orfiltration through successively smaller filter openings are preferred.The purpose is to remove all particles about 0.22 p. or larger, thusremoving all the bacteria present. The supernatant or filtrate isthereby sterilized to insure against bacterial contamination.

Test animals divided into appropriate groups are then immunized with theextracts and, after a suitable time interval, serum is obtained from theanimals. The presence of antibodies in the test sera is demonstrated byeither the Ouchterlony technique of double diffusion in agar gel,immunoelectrophoresis, hemagglutination reactions or passive cutaneousanaphylaxis. The preferred practical method, because of its simplicityand reproducible results is the Ouchterlony technique.

Once the antibodies are demonstrated to be present, it is possible todetermine if a particular extraction technique does, in fact, isolate afraction which contains carcinoembryonic antigen material, component Aor component B.

I have discovered an extraction and purification technique which finallyresults in two separate fractions each of which invariably produces oneprecipitant line in the Ouchterlony technique when tested againstnonabsorbed antisera. The technique, according to this invention, alsoprovides a means wherein CEA components A and B are separated from eachother and from materials of the same molecular weight and thus areisolated in substantially pure form.

CEA materials as well as components A and B are isolated and purified,according to the preferred process of this invention, from primary ormetastatic carcinoma tissue. Also, CEA material as well as components Aand B can be isolated and purified, according to the process of thisinvention, from embryonic digestive organs of fetuses in the second toseventh month of gestation and from meconiums. The following descriptionwill in most respects be directed to extraction from cancer tissue;however, the process may also apply to embryonic tissue or meconium.

CEA material, CEA component A or CEA component B in either embryonicdigestive organ tissue from the first and second trimester, meconium oradenocarcinoma tissue are extracted with a glycoprotein solvent in whichCEA material, component A and component B are soluble. This is requiredso that precipitable normal proteins and interfering antigenic materialscan be separated from the CEA material or components A and B.Glycoprotein solvents which are suitable are, e.g., perchloric acid,trichloroacetic acid, phosphotungstic acid and the like. However,perchloric acid, because of its availability and ease of use ispreferred.

Prior to the addition of the glycoprotein solvent, the material which isbeing treated is homogenized with water in order to solubilize the CEAmaterial or component A or component B, whichever is present. The amountof water should be sufficient to solubilize all of the carcinoembryonicantigen material or component A or component B. Generally, about twoliters of water per about every kilogram of treated material issufficient. More water can be used, however, it is usually notnecessary. It is preferred to use distilled water since the chances ofcontamination are thereby reduced. The homogenization can be carried outat from about 4 C. to about 60 (3., however, from about 4 C. to aboutroom temperature (about 20 C. to about 25 C.) is preferred.

The solid particles are then removed from the homogenate. Since the CEAmaterial, component A and component B are water soluble, this can beaccomplished by any convenient method of separation, e.g., filtration orcentrifugation and the like. Centrifugation is preferred because it isfaster and sufficient force can be developed to remove substantially allthe solid particles. Generally, about 3,000 to about 8,000 revolutionsper minute are sufficient to accomplish this. The separation ispreferably carried out at cold temperatures, e.g., about 4 C. to aboutC., to prevent loss of activity and at these temperatues the time forsedimentation may be decreased.

The supernatant from the centrifugation is then treated with aglycoprotein solvent to remove protein materials and interferingantigenic materials.

Any temperature below room temperature is suitable for the addition ofthe g'lycoprotein solvent to the supernatant of the homogenate.Preferably, however, from about 4 C. to about room temperature is used.The temperature of the glycoprotein solvent which is added to thesupernatant can also be variable, preferably, however, the sametemperature as the extracting temperature is utilized. Generally, aconcentrated acid is used as the glycoprotein solvent, e.g., about 0.5Nto about 2N with 2N being preferred. The solvent is added in about equalvolume to the supernatant. The time in which the reaction takes place isusually about 5 to about 30 minutes. Longer times are undesirable sincethey can result in loss of antigencity.

A precipitate results. This precipitate is separated from thesupernatant containing the dissolved CEA material, component A orcomponent B. Any convenient method of separation is suitable, e.g.,filtration, centrifugation and the like, however, centrifugation underthe same conditions as used with the homogenate is preferred.

Perchloric acid, salts such as sodium chloride and other low molecularweight materials are then removed in order to further purify the system.While it may be possible to accomplish this by precipitating theremaining proteins, I have discovered a fast, efficient methodcomprising dialysis through a semipermeable membrane against apolyethylene glycol with an average molecular weight of about 15,000 to20,000 and a softening point at 60 C. A typical suitable commercialproduct useful for this dialysis is 20 M Carbowax" marketed by MannResearch Laboratories. The dialysis is a critical part of the processsince it is fast and efficient and eliminates substantially alldiffusible soluble materials except the higher molecular weightmaterials which include the materials containing CEA activity. Thedialysis is carried out at 4 C. to 10 C., preferably 4 C. and iscompleted in about 18 hours. The process to this point takes about 24hours to complete.

The use of the 15,000 to 20,000 molecular weight polyethylene glycol inthe dialysis step is critical to this invention since it aids inspeeding up the isolation of the CEA material, component A or componentB, by the use of only one dialysis step rather than time consumingmultiple dialysis steps against water and eliminates the need forlyophylizing the retentate.

The resulting retentate is substantially solid in character and containsseveral materials having both higher and lower molecular weights thanthe CEA material, component A or component B.

The separation of the portion of the resulting retentate which containsthe CEA material, or component A or component B to the substantialexclusion of other materials is accomplished according to this inventionby sequential chromatography with two different gel columns followed bychromatography with an appropriate ion exchange column. The elutedfractions from the column chromatography which have a molecular weightof about 200,000-500,000 and a definite peak at the spectrophotometricabsorption wave length of 280 my. are those containing the CEA material,component A or component B.

The column chromatography can be accomplished by subjecting theretentate, in solution, to sequential chromatography on two differentgel columns in any order. Practically, however, when using carcinomatissue, a gel column which is used first in accordance with thisinvention is an agarose gel. Agarose is the neutral portion of agar. Thegel material is commercially available from AB Pharmacia, Uppsala,Sweden, under the trade name Sepharose. The gels are available asaqueous suspensions in 0.02 percent sodium azide as a preservative. Thegel structure is due to hydrogen bonding. The gel is prepared in beadedform having a selected particle size and percent agarose. Theconcentration of the agarose in the gel determines its fractionationrange.

The gels most suitable for use in this invention are those which have aparticle size of from 40 to 210 microns and contain 6 percent by weightagarose. These materials named Sepharose 6B have a fractionation rangewhich separates materials of molecular weight 4 X 10 or less. In theprocess of this invention, Sepharose 6B is used since, when carcinomatissue is used, it permits the separation of the fraction containing theCEA material or its components from extraneous materials ofsubstantially higher or lower molecular weight as well as from colloidalparticles.

The second column contains a gel filter material which is a hydrophilicwater-insoluble cross-linked dextran polymer gel. This material and themethod of its manufacture are described in British Pat. No. 854,715. Thegel material, which is commercially available from AB Pharmacia,Uppsala, Sweden, under the name Sephadex, comprises a three dimensionalmacroscopic network of dextran substances bonded or crossJinkedtogether, being capable of absorbing water with swelling. The ability ofthe gel material to take up water is inversely proportional to thedegree of crosslinkage of dextran substances in the gel material. Thegel material is available in a variety of grades differing with respectto degree of porosity. The gel preferred for use in this invention hasan approximate molecular weight exclusion limit of 100,000, a waterregain (g. H O/g. dry gel) of 10 i 1.0, a particle size of 40-120microns and a bed volume/ml./g. dry gel of l520. The gel is namedSephadex G-l00.

Sephadex G-100 is employed to further purify the fraction containing theCEA material or component A or component B. Since the columnhas greaterresolving power than the first column for the molecular weight range of100,000 to 200,000, further separation of the CEA material or componentA or component B from lower molecular weight materials is achieved. Thesecond column, for all practical purposes, should be used only after thecolloidal particles are removed by the first column since theseparticles will clog the column and make it ineffective. The problem ofcolloidal particles is applicable to the treatment of tumor tissue.However, when, for example, meconium is used, it is preferred to use theSephadex G-100 column first since it removes bile salts. After theremoval of the bile salts, then the Sepharose 68 column isadvantageously used.

The chromatography is accomplished by dissolving the retentate inaqueous buffer at a pH of from about to 9, preferably pH 7. A typicalsuitable buffer composition useful in the process of this invention iscomposed of 0.1 M Tris-OH adjusted to pH 7 with HCl made in 0.135 M NaClwith 0.02 percent of sodium azide as a preservative. The thus formedbuffer solution is then run through the first column, eluted with thesame buffer solvent and the eluates collected. The eluates are thendialyzed against the polyethylene glycol as described above. Thecollected active fraction is then redissolved in an aqueous Tris-OHbuffer of pH 5 to 9 of the same composition as described above, thesolution is run through the second column, eluted with a buffer of pH 5to 9 of the same composition as described above and the active fractionsare collected and dialyzed as before.

The advantage of utilizing low temperatures, i.e., from about 4 C. toabout 10 C. is that it maintains stability and can result in increasedresolution. The fractions collected from the second column are thosewhich have a molecular weight of 200,000-500,000 and have a reading witha peak at 280 my. on a UV spectrophotometer. Those fractions collectedfrom the first column are selected based on the same criteria, however,they contain material slightly greater and slightly less (as low as70,000 MW) than 200,000-5 00,000 MW. The collected fractions contain theCEA material or component A or component B depending on the origin ofthe treated material. This is shown by either the precipitin inhibitionor direct Ouchterlony testing against unabsorbed tumor antiserum. Asingle line precipitate indicates pure CEA activity.

The active fraction from the second gel column is then subjected tochromatography on an ion exchange column in order to further purify andfractionate the CEA active fraction and separate it from other materialswhich are present. I have found that in most cases the fractioncontaining the CEA activity which is derived from colon adenocarcinomatissue from the second gel column contains three different materials(unless, as in some cases of colon adenocarcinoma, only component A orcomponent B are present singly), all having molecular weights betweenabout 200,000 and 500,000. Of these materials, one comprising about 5percent by weight of the fraction is non-reactive. A second material,comprising about 10 percent by weight of the fraction has antigenicsites which react with the CEA specific antibody and is identified hereas CEA component B. A third material, comprising about percent by weightof the fraction also has antigenic sites which react with the CEAspecific antibody and is identified here as CEA component A.

It has also been found that other materials, e.g., meconium, lungtumors, breast tumors, have different proportions of the components andas a general rule these amounts vary from patient to patient and fromtumor to tumor. Meconium, for example, has only component B.

In order to obtain the pure CEA components it has been found necessaryto utilize an ion exchange column. If only one component is present,then the ion exchange column is used to purify it and confirm itspresence as the sole CEA active material present.

The ion exchange column found suitable for use in accomplishing thedesired separation is a mixed bed column composed of a cation exchanger,carboxymethyl cellulose, and an anion exchanger, diethylaminoethylcellulose.

The carboxymethyl celluloses most suitable for use in this invention arethose which are microgranular in form, have rod shaped particles with aparticle size distribution expressed as diameter of equivalent sphereswithin a range of about 20 ,u. to about 60 ,u., have a capacity of 1.0 i0.1 meq./gm. and a water regain of 2.3-2.7 gm./gm. dry exchanger. Thepreferred ionic form is the Na form. A suitable ion exchanger iscommercially available in a preswollen form from H. Reeve Angel Inc.,Clifton, N.J., under the trade name CM 52.

Another suitable carboxymethylcellulose is CM 32. This does not have thecapacity per volume of CM 52 but is otherwise similar, i.e., it ismicrogranular in form, has rod shaped particles with a particle sizedistribution expressed as diameter of equivalent spheres within a rangeof 20 p. to 60 ,u., has a capacity of 1.0 i 0.1 meq./gm. and a waterregain of 2.32.7 gm./gm. dry exchanger. CM 32 is available in dry fromfrom H. Reeve Angel lnc., Clifton, NJ.

The diethylaminoethylcelluloses most suitable for use in this inventionare those which are microgranular in form, have rod shaped particleswith a particle size distribution expressed as diameter of equivalentspheres within a range of about 20 p. to about 60 u, have a capacity of1.0 i 0.1 meq./gm., a water regain of 2.3-2.8 gm./gm. dry exchanger andare in the free base form. A suitable ion exchanger is commerciallyavailable from H. Reeve Angel Inc., Clifton, NJ. under the trade name DE52.

The mixed column is produced by removing the fines from each exchangerby, for example, aspiration of the supernatant resulting from adding a10-fold volume of water, stirring and allowing to settle. Subsequently,a solution made from ammonium acetate in 1.0 M sodium chloride is addedto each column and equal volumes of each of the resulting slurries arethen combined and poured in a 2.5 X 40 cm. column to give a 2.5 X 18 cm.mixed column.

The eluate from the gel columns is dialyzed against the polyethyleneglycol as described above. The resulting material is then dissolved inan aqueous buffered solvent which solubilizes proteins and does not haveaffinity for the column.

A typical suitable solvent is ammonium acetate at pH 4. The bufferedammonium acetate solvent can be formed by adjusting the pH of 0.1 Macetic acid with ammonium hydroxide.

The resulting solution is then clarified. The preferred clarificationmethod is centrifugation which effectively removes all the undissolvedparticles. High speed centrifugation is most effective for thisclarification, preferably at speeds which produce at least 100,000 timesgravity.

The resulting supernatant is then applied to the mixed bed ion exchangecolumn and eluted with an ammonium acetate-sodium chloride eluant at pH4. Other alkali metal chloride salts such as potassium chloride are alsosuitable. The relative amounts of ingredients in the eluant compositionsare varied. The variations in the composition results in a fineseparation of the active fraction into its major components and numerousminor components. This is accomplished by utilizing compositionscontaining the ammonium acetate solvent in 0.05, 0.1, 0.25 and 1.0 Msodium chloride solutions. The specific relationship of the ammoniumacetate to the sodium chloride is interrelated to the pH of the system,thus, if a different pH is utilized, then the relationship must bechanged to accomplish the same purpose. The identity and relativeamounts of the major components varies with the identity of theirsource. For example, in a typical case wherein colon carcinoma is thesource of antigen activity, about 85 percent by weight of the materialpresent in the active fraction is eluted when the eluant containsammonium acetate in 0.05 M sodium chloride. This is CEA component A.About 10 percent by weight of the material present in the activefraction is eluted when the eluant contains ammonium acetate in 0.1 Msodium chloride. This is CEA component B. The remaining material iseluted when the eluant contains ammonium acetate in 0.25 M sodiumchloride. In cases wherein only component A is present or only componentB is present, then the component present will be eluted with the eluantsas described for each component.

In addition to having the same electrophorectic characteristics as CEAmaterial, i.e., migrating anodally l0-l4 cm. in block electrophoresis atthe same time as ferritin marker migrates 18 cm. anodally, using 400volts and about 20mA with a borate buffer of pH 8.6 and ionic strength0.05, CEA component A has a molecular weight of between 120,000 and240,000, is eluted from a mixed bed ion exchange column having thecomposition as described with an ammonium acetate-sodium chloride eluantat pH 4 wherein the eluant contains ammonium acetate in 0.05 M sodiumchloride. Component A also forms a single line precipitate with itsspecific antibody in unabsorbed antiserum in gel diffusion tests, issoluble in perchloric acid and has a spectrophotometer absorption peakwave length of 280 mp" Also, CEA component B in addition to having thedescribed electrophoretic characteristics of CEA material has amolecular weight of between 120,000 and 240,000, is eluted from a mixedbed ion exchange column having the composition as described with anammonium acetate-sodium chloride eluant at pH 4 wherein the eluantcontains ammonium acetate in 0.1 M sodium chloride. Component B alsoforms a single line precipitate with its specific antibody in unabsorbedantiserum in gel diffusion tests, is soluble in perchloric acid and hasa spectrophotometer absorption peak wave length of 280 mu.

The material or components containing CEA activity are determined byeither the precipitin inhibition or direct Ouchterlony test againstunabsorbed tumor antiserum. A single line precipitate indicates pure CEAactivity. Thus, any material which forms a single line precipitate withunabsorbed CEA antiserum by either the precipitin inhibition or directOuchterlony technique of double diffusion in agar gel is included withinthe scope of this invention and is suitable for use in the diagnostictests described herein.

In order to utilize these techniques, the antibodies used must beconfirmed to be specific for CEA material, component A and/or componentB. Antibodies which meet this criteria can be produced by immunologicaltolerance or absorption techniques as described above.

Once the antibodies are demonstrated to be present, it is possible todetermine if a particular extraction technique does, in fact, isolatecarcinoembryonic antigen material, component A or component B. Usingthese techniques, I have found that when the CEA material is present,component A and component B respectively, obtained from the mixed bedion exchanger contains substantially all the CEA activity present in theCEA active fraction. The component which is preferred for use in theradioimmunoassay of CEA is component B. However, either the CEA material or component A can be satisfactorily utilized.

In another aspect of this invention, I have discovered radioimmunoassaytechniques which are simple to perform and have a high degree ofreproducibility and specificty.

In radioimmunoassays, it is important that the radioactive atom besufficiently reactive with the molecule to be tagged to provide anadequate concen tration of radioactivity for determination and theradioactive atom must provide a sufficient number of disintegrations perunit of time to provide sufficient sensitivity for accuratedeterminations. Further, in the case of radioimmunoassay of antigens,the antigenicity must not be deleteriously affected by the conjugationof the radioactive atom to the antigen.

By means of the present invention, it is possible to detect theexistence of human carcinoma growth by as saying a circulating tumorassociated antigen. This invention provides a test sufficientlysensitive to detect at least 1 ng. of CEA material, component A orcomponent B per ml. of serum or plasma. This sensitivity has been foundsufficient to detect abnormal amounts of CEA activity. A very minoramount, e.g., less than 0.05 ng. of CEA activity may be present innormal situations. The sensitivity of the assay is limited only by thespecific activity of the radioactive atom.

The CEA material, component A or component B can be tagged withradioactive atoms which will react with their chemically reactive groupsand not substantially diminish their antigenicity. I has been found tobe a particularly suitable radioactive atom.

The CEA material, component A or component B can be radio-iodinated bymethods known in the art, with minor modifications to concentration andvolumes. The Chloramine T method of Hunter and Greenwood, Biochem. J.91, 46 125 is particularly useful.

A radioiodination efficiency of about 20 percent to 50 percent can beobtained by the process described herein. The radioiodination process isequally applicable to the CEA material which is purified and isolatedprior to its fractionation into components A and B, or each of thecomponents. Preferred for use in this invention, however, is componentB.

The reaction is effected, for example, by using a 200 1. reactionmixture containing 100 ug. of Chloramine T (sodiump-toluenesulfo-chloramine); 0.0250.4 mg. of CEA material or anindividual component thereof and 4 mCi of I in the form of Kl or Nal.The reaction takes place in about 1 minute at room temperature and isstopped by the addition of sodium metabisulfite. The function of theChloramine T is to oxidize the iodide salt to iodine. The function ofthe sodium metabisulfite is to reduce unreacted I back to its salt.Other reducing agents can also be used, e.g., potassium metabisulfite.The oxidizing and reducing agents used should not be so strong that theydamage the antigenicity of CEA material or its components. Theradioiodinated product can be separated from residual unreacted l bychromatography in a cross-linked dextran gel column, e.g., Sephadex6-100, and removing the tube with the greatest radioactivity in thefirst peak. The resulting product has a specific acitivity of betweenabout l,000-25,000 dpm./ng., preferably between about 10,000 and 20,000dpm./ng., i.e., about 5-10 my. Ci/ng. of CEA material, component A orcomponent B.

It is necessary, in order to achieve success in the aforesaid diagnostictechnique, to treat the patients blood in a manner which will insurethat all the CEA material, component A or component B, to the exclusionof interfering materials, is in the finally used serum or plasma. Thiscan be accomplished by treating blood serum or plasma from the patientswith a glycoprotein solvent which solubilizes the CEA material,component A or component B, and then clarifying the resulting solution.It has been found that both serum and plasma from the blood of patientsare suitable for use in this process, however, plasma is preferred.

The glycoprotein solvent which has been found suitable for this processis perchloric acid. Perchloric acid of 1.2 M or a sufficient amount toprovide a concentration of about 0.6 M or less of perchloric acid is thepreferred solvent since it removes interfering substances, freesantigenic sites ad lowers ionic strength. The resulting solutioncontaining dissolved CEA and component A or component B, if any arepresent, is then clarified. The preferred clarification method is tocentrifuge, collect the supernatant and dialyze against distilled water,then against buffered water (pH 6-6.25, ammonium acetate with 0.01 Macetate). This usually takes about 6 to 10 hours. The dialysis residue(retentate) can then be dried by lyophylization, this is not essentialhowever. By using thismethod a purified extract containing greater thanabout 95 percent of the CEA material, component A or component Boriginally present is produced.

It is important to this process that the extract is treated as describedsince the glycoprotein solvent which solubilizes the CEA material,component A or 1964) using iodine component B in the initial stepdissociates any pre-existing CEA-anti-CEA complexes and activates theantigenic sites in the patients serum or plasma, enabling the recoveryof substantially all the CEA activity originally present. This providesa method for detecting CEA activity in patients with primary carcinomasand metastatic carcinomas of varying origin.

It is also possible in another aspect of the radioimmunoassay techniquesof this invention to add the antibody directly to the supernatantresulting from the glycoprotein solvent extract of the patients bloodserum or plasma. This eliminates the need for time consuming dialysisprocedures and provides a method for detecting CEA materials, componentA and/or component B in patients having carcinoma.

It is further possible in a preferred aspect of the radioimmunoassaytechniques of this invention to treat the blood serum or plasma bydiluting in such a manner that its ionic strength is reduced, then addthe antibody directly to the dilution.

The dilution can be accomplished by adding at least volumes of eitherwater or a salt solution of low ionic strength to each volume of theblood serum or plasma. It is preferred to use plasma. Generally anyconvenient salt can be used as long as it does not interfere with thesubsequent treatment with zirconyl phosphate. The salts found suitableare, for example, ammonium acetate, sodium chloride, sodium borate (pH8.4) and the like. Ammonium acetate of 0.01 M or less is preferred.

The dilution of the blood serum or plasma is for the purpose of loweringthe ionic strength of the solution in order to free or activateantigenic sites of any free CEA material or component which is present.This technique does not dissociate any pre-existing CEA- anti CEAcomplex but makes possible detection of free circulating CEA activity.It is important when using salt solutions as the diluent, that themolarity of the salt be sufficient to lower the ionic strength of theserum or plasma to a level which will activate the antigenic sites.

Since no dialysis procedures are required, this procedure savesconsiderable time and is suitable for initial screening procedures todetect free circulating CEA activity.

Further, in order to effectively conduct the radioimmunoassay, a supplyof antibodies specific to the CEA material, component A and/or componentB must be assured. This is accomplished by immunizing animals with thepurified CEA material or a component in conventional manner as follows.

An emulsifier, e.g., Freunds adjuvant (complete) is added to the CEAmaterial or either component in a saline solution. The emulsion can beinjected in animals intramuscularly, subcutaneously, in the foot pad orany combination of these methods. Animals such as fowl, rabbits, horses,goats, sheep and the like are suitable. The regimen in rabbits, forexample, is injections twice a week until five injections are made.After the last injection, blood is collected from the animal. The serumfrom this blood is unabsorbed CEA antiserum.

In one method, 400 pg. of CEA material or a component in 1 ml. salinesolution (0.9 percent) is utilized. The injection is madeintramuscularly using a volume about four times that injected in thefoot pad.

The antibody present in the antiserum, after absorption with normaltissue components, is specific in its activity against the CEA material,component A and/or component B to the exclusion of other antigens.

In conducting the radioimmunoassay of CEA, procedures based on both thetechniques of isotope dilution and competitive-inhibition can be used.However, the competitive-inhibition method is the preferred method ofthis invention. In these methods, a titration curve, then a standardinhibition curve are obtained.

The standard inhibition curve can be made by the Farr procedure. It is ameasure of the complex formation with specific antibodies. The curvereflects the amount of CEA material, component A and/or component Bpresent per unit of serum. The measurement is in nanograms per ml.,which is plotted against a known percentage of radioactive tagged CEAmaterial, component A or component B. The resulting curve is used toplot the amount of CEA material, component A or component B in apatients serum.

In a preferred method, a standard inhibition curve can also be obtainedby the competitive-inhibition method by adding standard CEA material,component A or component B to a series of tubes containing powderedperchloric acid extract of normal human serum or plasma. A measuredamount of CEA antiserum which had previously been determined from astandard dilution curve is added to the series of tubes containing adialyzed perchloric acid extract of normal blood serum or plasmadescribed above, or alternatively serum or plasma diluted with 0.01 M(0.01 N) ammonium acetate buffer at pH 66.25. In this alternative methodwherein the test fluid is diluted, a maximum normality of buffer shouldnot be greater than 0.01. Lower normalities are suitable. Whereappropriate, molarity can be used to describe the concentration,equivalent normalities can be calculated by conventional means.

The resulting solutions are incubated at about 45 C. for a sufficienttime to complete the reaction, usually about 30-45 minutes issufficient. Following the incubation, a measured amount ofradioiodinated CEA material, component A or component B is added to eachof the tubes. The incubation is then continued for about an additional30 minutes at about 45 C. When the incubation is completed, aprecipitant which precipitates the antibody and antigen-antibody complexbut not the antigen, is added to the solution to coprecipitate theantibody bound CEA material, component A or component B. Preferably, azirconyl phosphate gel is used.

Under the conditions described above, free CEA material, component A orcomponent B remains in solution, P content of the precipitate orsupernatant is then determined from a reading on a suitable instrumentand the amount of CEA material, component A or component B in the serumor plasma is then determined by reference to a standard.

The assay performed on the powdered perchloric acid extracts of serum orplasma processed in the same manner as the standard CEA material,component A or component B, results in a determination of the amount ofCEA material, component A or component B in the patients blood. This inturn is indicative of the presence or absence of carcinoma in thepatient.

According to this invention, the radioimmunoassay can be accomplished byeither a routine isotope dilution procedure or thecompetitive-inhibition assay method described above.

The isotope dilution assay method is carried out by adding a measuredamount of tagged CEA material, component A or component B to aperchloric acid extract of blood serum or plasma which is then dialyzed.The extract is then neutralized with, e.g., NaOl-l, and a measuredamount of antibody is added. The mixture is then dialyzed against thepolyethylene glycol described previously, driving the antibody-antigenreaction to completion.

The resulting precipitate is then dissolved in boric acid buffer at pH6.25. The radioactivity is then determined by adding zirconyl phosphategel to the solution, then centrifuging and assaying the precipitate forradioactivity.

The preferred competitive-inhibition assay method described above iscarried out by dissolving the solid perchloric acid blood serum orplasma extract in a suitable buffered solvent at a pH of 5-8, preferably6.25. While any conventional buffer is suitable, e.g., phosphate buffer,I have found that buffered solvents containing boric acid are preferred.This is surprising since heretofore borate buffers have been consideredunsuitable for use in radioimmunoassay or isotope dilution assay at anacid pH. The use of acidic conditions is dictated by the fact that theCEA material, component A or component B are not sufficiently stable atneutral or alkaline pHs to maintain their antigenicity.

A measured amount of antibody is then added to the solution. While anyamount is suitable, 30 units is used for convenience and ease ofmeasurement, however, from about 30 to about 300 units are suitable.

A unit of CEA activity is a nanogram of CEA material, or the equivalentamount of component A or component B. A unit of antibody is the amountof antibody which is bound by a nanogram of CEA material, or theequivalent amount of component A or component B.

The resulting mixture is then incubated for about 24 hours. 50 Units oftagged CEA material or the equivalent amount of component A or componentB are then added and the mixture is againincubated for about 24 hours.It is possible, however, to use from about 20 to about 500 units,however, 20-50 units have been found to be preferred. If there is someCEA material, component A or component B in the serum or plasma, thenthe amount of unreacted tagged CEA material, component A or component Bin the serum or plasma can be determined either qualitatively orquantitatively. The radioactivity is determined by adding zirconylphosphate gel to the solution, then centrifuging and assaying theprecipitate for radioactivity.

In another preferred aspect of this invention, the assay for determiningfree circulating CEA material, component A or component B is carried outby diluting either blood serum or plasma with at least volumes of wateror a low ionic salt solution as described previously.

The solution is then transferred into suitable test tubes, 30 units ofCEA antiserum are added and the mixture is incubated at about 45 C. for30-45 minutes. 20 to 50 Nanograms of radioiodinated CEA material orequivalent amounts of component A or component B, having 10,000 to20,000 dpm./ng. are then added and hours to complete. It is suitable fordetermining only the free CEA activity. When used in conjunction withthe competitive-inhibition assay methods, it is possible to have largescale screening for carcinomas.

The following examples illustrate the invention.

EXAMPLE 1 150 Grams of frozen primary colon adenocarcinoma tumor washomogenized in volumes of distilled water at 5 C. for 2 minutes in ahomogenizer. The homogenate was then blended for about 5 minutes in ablender. The resulting material was then centrifuged for 30 minutes at5,000 rpm. The supernatant was decanted and a stick was used to preventthe top fat pad which forms from breaking and contaminating thesolution. One volume of percent perchloric acid was added to thesupernatant and stirred at 5 C. for 10 minutes. The resulting mixturewas centrifuged for 30 minutes at 5,000 rpm. The supernatant wasdecanted and filtered through glass wool. The resulting filtrate wasthen dried by dialysis against a 20 M Carbowax solution which wasprepared by filling a 10 liter beaker with 20 M Carbowax Flakes andfilling it to the 7 liter mark with a borate buffer at pH 8.4, thenstirring until the flakes dissolved. The resulting solid dissolved in 8ml. of Tris(hydroxymethyl)-aminomethane-NaCl (Tris-NaCl) solution. Theresulting solution was centrifuged for 30 minutes at 105,000 gravity and5 ml. of the resulting supernatant was applied to a Sepharose 68 columnand eluted with the Tris-NaCl solution using 80 drops per tube collectedin 5 ml. fractions at the rate of 0.5 ml./minute. Tubes 45-57 werepooled and concentrated by dialysis against 20 M. Carbowax. Theresulting concentrate was then applied to a Sephadex G-l00 column. Thiswas eluted with the Tris-NaCl solution and 4 tubes containing 5 ml. eachof the first peak were pooled and dried by dialysis against 20 MCarbowax. The resulting solid material was dissolved in 2 ml. of theTris-NaCl solution and 1 ml. was labelled with l by conventional means.The I labelled material was applied to a Sepharose 68 column and elutedwith the Tris-NaCl solution. The pooled fractions 45-57 were frozen in 5ml. tubes and stored at -20 C. This is called Tumor Extract No. l(TE-l). When studied by gel diffusion versus goat antiserum, a singlestrong band appeared. In certain tumor extracts a second minor bandappeared.

2 Ml. of labelled TE-l were applied to a CM-52zDE- 52 column in 50 ml.of ammonium acetate (pH 4) solution and the column was washed with 150ml. of ammonium acetate. Almost all the P was retained by the mixedcellulose ion exchange column. The column was then eluted with 500 ml.each of ammonium acetate- NaCl solutions containing 0.05 M NaCl, 0.1 MNaCl, 0.25 M NaCl, 1.0 M NaCl. Two peaks were eluted with the 0.05 MNaCl-buffer. The first peak is that of CEA component A. The second peakappeared to be degraded 120,000 molecular weight material which iscalled M- l 20. A second major peak was eluted with the 0.1 MNaCl-buffer and is a pure material having 240,000 molecular weight, itis CEA-component B. A third major peak was eluted with the 0.1 MNaCl-buffer and was not reactive with the antisera. This indicates it isprobably a normal component. Thus, the first peak which was eluted withthe 0.05 M NaCl is CEA component A which contains CEA activity. Thesecond major peak, CEA component B, also contains the CEA activity. Theidentity of the CEA material, component A and component B is confirmedby its forming a single line in the Ouchterlony gel diffusion test withunabsorbed anti-serum. When subjected to block electrophoresis usingSephadex G-25 Fine [a cross linked dextran gel having an approximatemolecular weight exclusion limit of 5,000, a water regain (gl-l Olg. drygel) of 2.5 i 0.2, particle size of 20-80 microns and a bedvolume/ml./g. dry gel of 5] on a non-conductive block, e.g., Lucite, theCEA material, component A and component B behave identically as follows:

The block electrophoresis medium, Sephadex G-25 Fine is swollen withwater for 2 hours at C. and washed by decantation with borate of pH 8.6and ionic strength 0.05. then suction filtered through a sintered glassdisk.

A thick slurry of the gel is poured onto a Lucite block support of 61cm. X 7.5 cm. X 1 cm. in dimensions and allowed to distribute itselfevenly along the plate to a depth of 1 cm. The surface is then blottedwith cotton gauze sponges until firm but not completely dry.

The block is then fitted with 3 mm. chromatography paper contacts(Whatman) all aligned in the same direction of flow of the paper. Theblock is then placed in the electrophoresis apparatus and allowed toequilibrate for 1 hour under the operating conditions of 400 volts, witha constant current of approximately 20 mA at 4 C. A 1 cm. strip is thenremoved from the center of the block and mixed well with a solution of60 mg. of CEA material produced as above, in 0.5 ml. of 0.05 M borate.The resulting slurry is then poured back in the central strip. One totwo drops of ferritin 6 X recrystallized) at a concentration of mg./ml.is then spotted at the cathodal extremity of the block. 24 Hours afterthe start of the run, the ferritin marker moves 18 cm. anodally. At thattime the block is removed from the electrophoresis apparatus and 2centimeter strips between the zone of application and the anodalextremity are eluted with 2 M NaCl passed through 0.20 p. disposablegrid membrane (Nalgene). The activity is localized 10-14 cm. anodal tothe application zone with weaker activity being found 8l0 cm. anodal tothe application zone.

When components A and B are treated separately in the same manner,identical results are obtained.

EXAMPLE 2 6 ml. tubes of normal serum and six 5 ml. tubes of serum fromsuspected colon cancer patients each were extracted with an equal volumeof 1.2 to 2 molar perchloric acid by shaking for 20 minutes and thencentrifuging at 8,000 gravity for 5 minutes at 5 C. The supernatantswere collected and transferred to a dialysis tubing and placed in a 250ml. beaker of a Carbowax solution formed by filling a l. beaker with 20M Carbowax Flakes and then filling the beaker to the 7 liter mark withborate buffer of pH 8.4 and stirring until the flakes dissolve. After 5hours of dialysis, the resulting precipitate in the tubes was dissolvedin 1 ml. of borate buffer at pH 6.25 and transferred to X 125 mm. testtubes. 0.1 Ml. of normal human serum was added to each tube and mixed.To each of the six tubes containing normal serum, perchloric acidextracts, 0, 10, 50, 100, 250 and 500 nanograms of CEA was added. Then300 to 500 units of CEA antisera was added to each of the 12 tubes andmixed. The tubes were then stored in an icebox at 5 C. for 12 hours.Subsequently, 500 units of CEA-l was added to each tube and incubationwas continued for 18 hours at 5 C. Five ml. of zirconyl phosphate gelwas added to the tubes and the tubes were then filled with ammoniumacetate buffer at pH 6.25. The tubes were stopped with rubber stoppers,inverted five times and centrifuged at 1,500 gravity for 5 minutes. Theresulting supernatant was then discarded. The solid gel which remainedwas washed with an ammonium acetate buffer by filling the tubes with thebuffer and dispersing the gel with a mixer, then centrifuging at 1500times gravity for 5 minutes. The gel was assayed for bound 1' with aPackard 3003 Tricarb Scintillation Spectrometer. Other similar equipment can also assay for the bound 1 The results of the serum beingtested for CEA activity were compared to the standard and the amount ofCEA material, component A or component B in the unknown sera wasdetermined.

EXAMPLE 3 0, 10, 50, 100 and 500 Nanograms of CEA material standard wereadded to separate tubes each of which contained 5 ml. of normal sera andthen mixed. The standards and serum from suspected cancer patients wereextracted with perchloric acid, centrifuged and dialyzed against MCarbowax in the same manner as in Example 2. The resulting precipitatewas dissolved in 1.0 ml. of borate buffer of pH 6.25, then 500 units ofradioactive tagged CEA material was added, the mixture was mixedthoroughly and then 300 units of CEA antisera were added. The mixturewas dialyzed against a fresh 20 M Carbowax solution and brought todryness in about 2 to 3 hours. The resulting precipitate was dissolvedin 1 ml. of borate buffer of pH 6.25 and then 5 ml. of zirconylphosphate gel were added. The assay for the l was made according to theprocess set forth in Example 2.

EXAMPLE 4 Human meconium is homogenized in 3 volumes of 10 percentperchloric acid at 5 C. and centrifuged at 4,000 rpm for minutes. Thesupernatant is then dialyzed against 20 M Carbowax.

The precipitate is taken up in a minimum volume of Tris-NaCl solution ofpH 7 and centrifuged at 105,000 g. for 30 minutes.

5 M1. of the supernatant is then applied to a Sephadex G-100 column andeluted with Tris-NaCl solution. Four tubes of 5 ml. each from the firstfraction were pooled and brought to dryness by dialysis against 20 MCarbowax. The residue is taken up in 8 ml. of NaCl-Tris solution andcentrifuged at 105,000 g. for 30 minutes. 5 M1. of the resultingsupernatant is then ap- EXAMPLE 5 Carcinoembryonic antigen (CEA)material was isolated and radiolabelled with I as described in Examplel.

A goat. antiserum mono-specific for CEA material was reacted withradiolabelled CEA material to form an antibody-antigen complex. Theexcess radiolabelled CEA material was separated from the complex byadsorbing the complex with zirconyl phosphate gel (pH 6.25) as describedin Examples 2 and 3.

The radiolabelled CEA was then incubated with the antiserum as follows:

Ng. of radioiodinated CEA material was incubated with antiserum dilutedwith water (ll0,000) at 45 C. for 30 minutes in 1 ml. each of normalserum (goat, human, rat, rabbit), 0.15 M NaCl, 0.075 M Na HPO 0.15 MTris-HCl (pH 7.5) and 0.1 M ammonium acetate. This resulted in minimalcomplex formation.

When the radioiodinated CEA material and the antiserum were incubated in1 ml. each of H O,0.0l M NaCl, 0.01 M ammonium acetate, 1 percent normalserum diluted in H O, or 0.05 M sodium borate (pH 8.4) antigen-antibodycomplex formation took place.

The antiserum also formed a complex with the radioiodinated CEA materialwhen incubated in 10 ml. of 0.01 M ammonium acetate, 0.1 ml. of normalserum diluted to 10 ml. with water, or 0.005 M sodium borate (pH 8.4).

10 Ng. of CEA material added to dialyzed supernatant from 5 ml. ofnormal serum and 5 ml. of 1 M perchloric acid, neutralized 10 percent ofthe antiserum when incubated at 45 C. for 30 minutes prior to theaddition of measured amounts of radioiodinated CEA material.

CEA material was detected in 28 of 30 perchloric acid extracts of serumobtained from patients with colon adenocarcinoma and directly in sera ofmetastatic patients after dilution of 0.1 ml. serum in 10 ml. of water.This indicates that dilution which weakens the ionic strength of theserum provides access to an antigenic site on the CEA material.

EXAMPLE 6 A 3 ml. aliquot of 2 M perchloric acid was added to 5 ml.aliquots of serum or plasma while agitating in a mixer. The mixtureswere allowed to stand at room temperature for 15 minutes then mixedagain and a1- lowed to settle. The mixtures were centrifuged at 1,000times g. for 5 minutes at room temperature and the supematants weredialyzed for 36 hours against 25 liters of distilled water at roomtemperature. The dialysis bath was changed five times during a 24 hourperiod. This retentate was then used for testing. All specimens were runin duplicate.

Goat antisera monospecific for CEA material was diluted 1:2000 in 10percent normal human serum and 0.05 M borate buffer pH 8.4. CEA materialwas prepared and labelled with l as described in Example 1.

A dilution curve of the antisera against a constant amount ofradioiodinated CEA material was carried out in the dialysates ofperchloric acid serum extracts to which 1 ml. of borate buffer (0.05 M,pH 8.4) was added.

Six tubes of the serum were placed in a water bath at 45 C. for VA hour.After incubation of the mixture, 5 ml. of ammonium acetate solution (0.1M, pH 6.25) prepared by adjusting the pH of 0.1 M acetic acid to 6.25with cone NH OH, and 4 ml. of zirconyl phosphate gel (pH 6.25) wereadded to each tube and the tubes capped and inverted several times. Thetubes were then centrifuged at 3,000 rpm for 15 minutes and thesupernatants were discarded. The residue from each tube was resuspendedin ml. of ammonium acetate solution, recovered by centrifugation andassayed for bound l in a gamma scintillation counter.

A titration curve was carried out by adding known amounts of unlabelledCEA material to the retentates of serum perchloric acid extracts. 1 M1.borate buffer (pH 8.4, 0.5 M) and 0.5 ml. of a 1:1000 dilution ofantisera was added to each of 6 tubes and incubated in a water bath at45 C. for /2 hour. Then 0.1 ml. of radioiodinated CEA materialcontaining 24,000 DPM was added to each specimen. The tubes were mixedwell and reincubated for one half hour. 5 M1. of ammonium acetatesolution (0.1 M pH 6.25) and zirconyl phosphate gel (pH 6.25) were thenadded to each tube. The tubes were capped, inverted several times andcentrifuged at 1,200 times g. for minutes at room temperature. Thesupernatant was discarded and the gel precipitate was resuspended in 10ml. ammonium acetate solution (0.1 M, pH 6.25). The gel was separated bycentrifugation and assayed for bound I Specimens from patients were runin exactly the same manner as above except that unlabelled antigen wasnot added.

About 75 percent of the 1 labelled material reacted with the antisera. Afinal dilution of 1:10,000 of the CEA-antisera in the perchloric acidextracts from serum reacts with 70 percent of the maximum amount oflabelled antibody reactive material. The 1:10,000 dilution was thereforeutilized.

Incubation of the antisera diluted with water (1110,000) with unlabelledCEA material prior to addition of labelled antigen shows that thereaction of antibody with antigen is linear at antigen concentrations offrom 1.5 to 10 ng./ml. but that the reaction is less sensitive atconcentrations above ng./ml.

Of 487 patients tested, those with carcinomas of the breast, lung andcolon had detectable concentrations of CEA material in their serum.

Of 229 patients without malignant disease, 11 had detectable antigen intheir serum. Two of these later developed cancer, one had adenomatiouspolyp of the colon and 5 had severe emphysema.

EXAMPLE 7 1 M1. of plasma from suspected cancer patients was dilutedwith 4 ml. of physiological saline solution. An equal volume of 1.2molar perchloric acid was added and the mixture was agitated for 20minutes then centrifuged at 8,000 times gravity for 5 minutes at roomtemperature. The supernatant was collected and transferred to a dialysistubing and dialyzed overnight against distilled water. The resultingretentate was dialyzed against an ammonium acetate solution of pH 6 to6.25 containing 0.01 molar acetate, for 3 hours at room temperature. Theretentate was transferred into 20 ml. test tubes. Units of CEA antiserumwas then added and the mixture incubated for 30 to 45 minutes at 45 C.50 Ng. of CEA-I containing 10,000 to 20,000 dpm/ng was then added andthe mixture incubated for 30 minutes at 45 C. 5 M1. of pH 6.25 zirconylphosphate gel was added to each test tube and 5 ml. of ammonium acetatesolution (pH 6.25, 0.1M) were then added. After mixing the tubes werecentrifuged at 1,500 times gravity for 5 minutes at room temperature andthe resulting supernatant was discarded. The solid gel which remainedwas washed with the ammonium acetate buffer by filling the tubes withthe buffer and dispersing the gel with a mixer, then centrifuging at1,500 times gravity for 5 minutes. The gel was assayed for bound 1 witha Packard 3003 Tri-carb Scintillation Spectrometer. If CEA is present inthe plasma, the

amount of bound CEA-1"" will be reduced proportionately.

EXAMPLE 8 10 Ml. of water were added to 0.1 ml. of plasma in a 20 ml.test tube. 30 Units of CEA antiserum were then added and the mixtureincubated for 30-45 minutes at 45 C. 50 Ng. of CEA-l containing 10,000to 20,000 dpm/ng. were then added and the mixture incubated for30minutes at 45 C. 5 M1. of pH 6.25 zirconyl phosphate gel and 5 ml. ofammonium acetate solution (pH 6.25, 0.01 M) were then added to each testtube. After mixing, the tubes were centrifuged at 1,500 times gravityfor 5 minutes at room temperature and the resulting supernatant wasdiscarded. The solid gel which remained was washed with the ammoniumacetate buffer by filling the tubes with the buffer and dispersing thegel with a mixer, then centrifuging at 1,500 times gravity for 5minutes. The gel was assayed for bound I with a Packard 3003 Tri-carbScintillation Spectrometer. If CEA is present in the plasma, the amountof bound CEA-l will be reduced accordingly.

What is claimed is:

1. A method for detecting the presence of carcinoembryonic antigenmaterial, component A and/or component B in blood which comprises a.adding a measured amount of antibody to a solution of a perchloric acidextract of blood serum or blood plasma in a buffered solvent at pH 6.25of a maximum normality of 0.01:

. incubating the mixture;

c. adding a measured amount of radioactive tagged carcinoembryonicantigen material, component A or component B to the incubated mixture;

. incubating the resulting mixture;

e. adding a precipitating agent to the incubated mixture therebyprecipitating all the CEA-anti CEA complexes; and

f. measuring the precipitate.

2. The method of claim 1 wherein in step (a) the buffer is a boratebuffer.

radioactive content of the 3. The method of claim 1 wherein in step (a)the buffer is ammonium acetate.

4. The method of claim 1 wherein in step (a) a perchloric acid extractof blood plasma is used.

5. The method of claim 1 wherein in step (c) the amount of CEA material,component A or component B added is about 50 nanograms.

6. The method of claim 1 wherein the in step (e) the precipitating agentis zirconyl phosphate gel.

7. The method of claim 1 wherein in step (c) radioactive tagged CEAcomponent A is added.

8. The method of claim 1 wherein in step (c) radioactive tagged CEAcomponent B is added.

9. The method of claim 1 wherein in step (c) radioactive tagged CEAmaterial is added.

10. A method for detecting the presence of carcinoembryonic antigenmaterial, CEA component A and/or CEA component B in blood whichcomprises a. adding a measured amount of tagged CEA material, componentA or component B to a perchloric acid extract of blood serum or bloodplasma prior to dialysis;

b. dialyzing the mixture against a polyethylene glycol with an averagemolecular weight of about 15,000 to 20,000 and a softening point ofabout 60 C.;

c. adjusting the pH of the retentate to about pH 6-7;

d. adding a measured amount of antibody to the adjusted mixture;

e. dialyzing against polyethylene glycol of molecular weight of about15,000 to 20,000 and having a softening point at 60 C.;

f. dissolving the resulting retentate in borate buffer of g. adding aprecipitating agent to the solution thereby precipitating all theCEA-anti CEA complcxes; and

h. measuring the radioactive content of the precipitate.

11. The method of claim 10 wherein in step (c) the pH of the mixture isadjusted by a borate buffer of pH 6.25.

12. The method of claim 10 wherein in step (f) the precipitating agentis zirconyl phosphate gel.

13. The method of claim 10 wherein in step (a) 1 tagged CEA material isused.

14. The method of claim 10 wherein in step (a)l tagged CEA component Ais used.

15. The method of claim 10 wherein in step (a) 1 tagged CEA component Bis used.

16. The method of claim 10 wherein in step (a) blood plasma is used.

17. A method for detecting the presence of carcinoembryonic antigenmaterial, component A and/or component B in blood which comprises a.diluting blood serum or blood plasma with at least 100 volumes of water;

b. adding a measured amount of CEA antiserum to the dilution;

c. incubating the mixture;

d. adding a measured amount of radioactive tagged carcinoembryonicantigen material, component A or component B to the incubated mixture;

e. incubating the mixture;

f. adding a precipitating agent to the incubating mixture therebyprecipitating all the CEA-anti CEA complexes; and g. measuring theradioactive content of the exchanger, and microgranulardiethylaminoethyl cellulose, in free base form, having rod shapedparticles, a particle size distribution with a range of about 20 p. toabout 60 u, a capacity of l.0 i 0.1 meq./gm. and a water regain of2.3-2.8 gm./gm. dry exchanger with ammonium acetate at pH 4 in 0.05 Msodium chloride, forming a single line precipitate with its specificantibody in unabsorbed antiserum in gel diffusion tests, being solublein perchloric acid, having a spectrophotometer absorption peak wavelength of 280 mp. and having a specific activity of 5-10 mu Ci/ng. ofcarcinoembryonic antigen component A.

20. Radioiodinated carcinoembryonic antigen component B characterized byhaving a molecular weight of between 120,000 and 240,000 being elutablefrom a mixed bed ion exchange column comprising an equal mixture byweight of either preswollen or dry microgranular carboxymethyl cellulosehaving rod shaped particles with a particle size distribution with arange of about 20 p. to about 60 ,u, a capacity of 1.0 :t 0.1 meq./gm.and a water regain of 2.3-2.7 gm./gm. dry exchanger, and microgranulardiethylaminoethyl cellulose, in free base form, having rod shapedparticles, a particle size distribution with a range of about 20 u toabout 60 pt, a capacity of 1.0 :t: 0.1 meq./gm. and a water regain of2.3-2.8 gm./gm. dry exchanger with ammonium acetate at pH 4 in 0.1 MNaCl, forming a single line precipitate with its specific antibody inunabsorbed antiserum in gel diffusion tests, being soluble in perchloricacid, having a spectrophotometer absorption peak wave length of 280 myand having a specific activity of 5-10 mp. Ci/ng. of carcinoembryonicantigen component B.

UNITED STATES PATENT UFFICE C ERTEFEQATE CF @oRt Patent NO. 'D oc tob erInventor(s) Hans John -Hans'en It is certified vtha'terror appears inthe above-identified patent and that said Letters Patent are herebycorrected as shown below:'

Claim 1, Column 20, line 5 L, after "pH" insert from 6 to Claim 10,Column 21, line 19, after "amount of" insert radioactive.

Signed and sealed this 10th day of July 1973.

SEAL) Attest:

EDWARD M.FLETCHER,JR. r Rene Tegtmeyer Attesting Officer ActingCommissioner of Patents FORM PC4050 (10-59) uscoMM-oc 60376-P69 A U.S.GOVERNMENT PRINTING OFFICE 2 1959 0-355-33L UNITED STATES PATENT OFFICEOF CORREQTION Patent No. 5, 97j 5 f h d' I October -10,

Inventor(s) Hans John Han-sen It is certified tha'terror appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:'

Claim 1, Column 20, line 54, after "pH" insert from 6 to Claim 10,Column 21, line 19,. after "amount of" insert az'adioactivev Signed andsealed this 10th day of July 1973.

(SEAL) Attest I EDWARD M.FLETCHER,JR. Rene Tegtmeyer Attesting Officer I7 Acting Commissioner of Patents F ORM PO-105O (10-69) USCOMM-DC60376-P69 u.s. GOVERNMENT PRINTING OFFICE I969 0-366-334,

2. The method of claim 1 wherein in step (a) the buffer is a boratebuffer.
 3. The method of claim 1 wherein in step (a) the buffer isammonium acetate.
 4. The method of claim 1 wherein in step (a) aperchloric acid extract of blood plasma is used.
 5. The method of claim1 wherein in step (c) the amount of CEA material, component A orcomponent B added is about 50 nanograms.
 6. The method of claim 1wherein the in step (e) the precipitating agent is zirconyl phosphategel.
 7. The method of claim 1 wherein in step (c) radioactive tagged CEAcomponent A is added.
 8. The method of claim 1 wherein in step (c)radioactive tagged CEA component B is added.
 9. The method of claim 1wherein in step (c) radioactive tagged CEA material is added.
 10. Amethod for detecting the presence of carcinoembryonic antigen material,CEA component A and/or CEA component B in blood which comprises a.adding a measured amount of tagged CEA material, component A orcomponent B to a perchloric acid extract of blood serum or blood plasmaprior to dialysis; b. dialyzing the mixture against a polyethyleneglycol with an average molecular weight of about 15,000 to 20,000 and asoftening point of about 60* C.; c. adjusting the pH of the retentate toabout pH 6-7; d. adding a measured amount of antibody to the adjustedmixture; e. dialyzing against polyethylene glycol of molecular weight ofabout 15,000 to 20,000 and having a softening point at 60* C.; f.dissolving the resulting retentate in borate buffer of pH 6.25; g.adding a precipitating agent to the solution thereby precipitating allthe CEA-anti CEA complexes; and h. measuring the radioactive content ofthe precipitate.
 11. The method of claim 10 wherein in step (c) the pHof the mixture is adjusted by a borate buffer of pH 6.25.
 12. The methodof claim 10 wherein in step (f) the precipitating agent is zirconylphosphate gel.
 13. The method of claim 10 wherein in step (a) I125tagged CEA material is used.
 14. The method of claim 10 wherein in step(a)I125 tagged CEA component A is used.
 15. The method of claim 10wherein in step (a) I125 tagged CEA component B is used.
 16. The methodof claim 10 wherein in step (a) blood plasma is used.
 17. A method fordetecting the presence of carcinoembryonic antigen material, component Aand/or component B in blood which comprises a. diluting blood serum orblood plasma with at least 100 volumes of water; b. adding a measuredamount of CEA antiserum to the dilution; c. incubating the mixture; d.adding a measured amount of radioactive tagged carcinoembryonic antigenmaterial, component A or component B to the incubated mixture; e.incubating the mixture; f. adding a precipitating agent to theincubating mixture thereby precipitating all the CEA-anti CEA complexes;and g. measuring the radioactive content of the precipitate.
 18. Themethod of claim 17 wherein in step (f) the precipitating agent iszirconyl phosphate gel.
 19. Radioiodinated carcinoemryonic antigencomponent A characterized by having a molecular weight of between120,000 and 240,000, being elutable from a mixed bed ion exchange columncomprising an equal mixture by weight of either preswollen or drymicrogranular carboxymethyl cellulose having rod shaped particles with aparticle size distribution with a range of about 20 Mu to about 60 Mu ,a capacity of 1.0 + or - 0.1 meq./gm. and a water regain of 2.3-2.7gm./gm. dry exchanger, and microgranular diethylaminoethyl cellulose, infree base form, having rod shaped particles, a particle sizedistribution with a range of about 20 Mu to about 60 Mu , a capacity of1.0 + or - 0.1 meq./gm. and a water regain of 2.3-2.8 gm./gm. dryexchanger with ammonium acetate at pH 4 in 0.05 M sodium chloride,forming a single line precipitate with its specific antibody inunabsorbed antiserum in gel diffusion tests, being soluble in perchloricacid, having a spectrophotometer absorption peak wave length of 280 m Muand having a specific activity of 5-10 m Mu Ci/ng. of carcinoembryonicantigen component A.
 20. Radioiodinated carcinoembryonic antigencomponent B characterized by having a molecular weight of between120,000 and 240,000 being elutable from a mixed bed ion exchange columncomprising an equal mixture by weight of either preswollen or drymicrogranular carboxymethyl cellulose having rod shaped particles with aparticle size distribution with a range of about 20 Mu to about 60 Mu ,a capacity of 1.0 + or - 0.1 meq./gm. and a water regain of 2.3-2.7gm./gm. dry exchanger, and microgranular diethylaminoethyl cellulose, infree base form, having rod shaped particles, a particle sizedistribution with a range of about 20 Mu to about 60 Mu , a capacity of1.0 + or - 0.1 meq./gm. and a water regain of 2.3-2.8 gm./gm. dryexchanger With ammonium acetate at pH 4 in 0.1 M NaCl, forming a singleline precipitate with its specific antibody in unabsorbed antiserum ingel diffusion tests, being soluble in perchloric acid, having aspectrophotometer absorption peak wave length of 280 m Mu and having aspecific activity of 5-10 m Mu Ci/ng. of carcinoembryonic antigencomponent B.